PROJECT SUMMARY Tau is a microtubule-associated-protein that is sorted into neuronal axons under normal physiological conditions. However, in Alzheimer's disease (AD) and other neurodegenerative tauopathies, pathological accumulation of tau occurs in somatodendritic compartment of neurons, which is widely considered to be an early pathological event that leads to synaptic dysfunction and degeneration. Extensive mouse genetic and cell biological studies also reveal that tau serves as an essential mediator of amyloid ?-peptide (A?)-induced synaptotoxicity. A? or pathogenic tau gene mutations trigger tau hyperphosphorylation and subsequently disrupts polarized distribution of tau. This results in somatodendritic accumulation (tau missorting phenotype) and axonal transport defects, which is perceived to be an early pathological change leading to the AD-relevant neuronal atrophy. Despite strong disease-relevance, there has been no large-scale, unbiased screening effort to discover novel pharmacological agents that could ameliorate these tau-associated neuronal alterations. One major challenge in the field has been the lack of a scalable physiological neuron model that recapitulates polarized tau distribution, and is also suitable for a reproducible cell-based compound screen. Our previous work describes a robust neuronal model system based on mouse embryonic stem cell-derived neurons (ESNs) and their use in AD-relevant cell-based assays including A?-induced synaptotoxicity. We found that our ESN model system produces highly robust pyramidal cell-like neurons with polarized distribution of subcellular dendritic and axonal markers. Treatment of the ESNs with A? oligomers resulted in profound re-distribution of tau into MAP2-positive somatodendritic compartments. This A?-triggered tau mislocalization phenomenon was recapitulated and quantifiable in a miniaturized 384-well plate format, which is considered to be ideal for a chemical biology approach. With this approach we will assess which cellular targets are involved in regulating A?-induced tau missorting by conducting small molecule screening of a bioactive compound collection with annotated target profiles or clinical drugs, followed by uncovering the underlying biological mechanisms. Based on our preliminary data, the overall goals of the current study are to establish and optimize a neuronal assay for A?-induced tau missorting and to conduct small molecule screening to discover pharmacological agents that can reverse the tau missorting phenotype. The successful completion of our proposed study will yield a number of bioactive compounds with known cellular targets, which are ideal for identifying cellular factors and pathways that are critically involved with A?-associated tau missorting. A subset of the identified compounds may potentially have translational value, especially those previously developed as medications for other disease indications (e.g. drug repurposing candidates), and may serve as a foundation for further therapeutic target biology and future drug discovery projects.